Paul Colville-Nash

Inducible cyclooxygenase may have anti-inflammatory properties

Cyclooxygenase (COX) has two isoforms. Generally, COX 1 is constitutively expressed in most tissues, where it maintains physiological processes; inducible COX 2 is considered a pro-inflammatory enzyme and a chief target for the treatment of inflammatory diseases. Here we present evidence that COX 2 may have anti-inflammatory properties. In carrageenin-induced pleurisy in rats, the predominant cells at 2 hours are polymorphonuclear leucocytes, whereas mononuclear cells dominate from 24 hours until resolution at 48 hours. In this model, COX 2 protein expression peaked initially at 2 hours, associated with maximal prostaglandin E2 synthesis. However, at 48 hours there was a second increase in COX 2 expression, 350% greater than that at 2 hours. Paradoxically, this coincided with inflammatory resolution and was associated with minimal prostaglandin E2 synthesis. In contrast, levels of prostaglandin D2, and 15deoxyΔprostaglandin J2 were high at 2 hours, decreased as inflammation increased, but were increased again at 48 hours. The selective COX 2 inhibitor NS-398 and the dual COX 1/COX 2 inhibitor indomethacin inhibited inflammation at 2 hours but significantly exacerbated inflammation at 48 hours. This exacerbation was associated with reduced exudate prostaglandin D2 and 15deoxyΔprostaglandin J2 concentrations, and was reversed by replacement of these prostaglandins. Thus, COX 2 may be pro-inflammatory during the early phase of a carrageenin-induced pleurisy, dominated by polymorphonuclear leucocytes, but may aid resolution at the later, mononuclear cell-dominated phase by generating an alternative set of anti-inflammatory prostaglandins.

Resolution-phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP

Neutralizing injurious stimuli, proinflammatory mediator catabolism, and polymorphonuclear leukocyte (PMN) clearance are determinants of inflammatory resolution. To this, we recently added innate-type lymphocyte repopulation as being central for restoring postinflammation tissue homeostasis with a role in controlling innate immune–mediated responses to secondary infection. However, although macrophages dominate resolution, their phenotype and role in restoring tissue physiology once inflammation abates are unknown. Therefore, we isolated macrophages from the resolving phase of acute inflammation and found that compared with classically activated proinflammatory M1 cells, resolution-phase macrophages (rMs) possess weaker bactericidal properties and express an alternatively activated phenotype but with elevated markers of M1 cells including inducible cyclooxygenase (COX 2) and nitric oxide synthase (iNOS). This phenotype is controlled by cAMP, which, when inhibited, transforms rM to M1 cells. Conversely, elevating cAMP in M1 cells transforms them to rMs, with implications for cAMP in the resolution of systemic inflammation. It transpires that although rMs are dispensable for clearing PMNs during self-limiting inflammation, they are essential for signaling postresolution lymphocyte repopulation via COX 2 lipids. Thus, rM macrophages are neither classically nor alternatively activated but a hybrid of both, with a role in mediating postresolution innate-lymphocyte repopulation and restoring tissue homeostasis.

The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFbeta1-Smad (transforming growth factor-beta1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFbeta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFbeta1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, alpha-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFbeta1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGFbeta1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFbeta1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

Dichotomy in duration and severity of acute inflammatory responses in humans arising from differentially expressed proresolution pathways

Lipoxins (Lxs) and aspirin-triggered epi-Lxs (15-epi-LxA4) act through the ALX/FPRL1 receptor to block leukocyte trafficking, dampen cytokine/chemokine synthesis, and enhance phagocytic clearance of apoptotic leukocytes—key requisites for inflammatory resolution. Although studies using primarily inbred rodents have highlighted resolution as an active event, little is known about the role resolution pathways play in controlling the duration/profile of inflammatory responses in humans. To examine this, we found two types of responders to cantharidin-induced skin blisters in male healthy volunteers: those with immediate leukocyte accumulation and cytokine/chemokine synthesis followed by early resolution and a second group whose inflammation increased gradually over time followed by delayed resolution. In early resolvers, blister 15-epi-LxA4 and leukocyte ALX were low, but increased as inflammation abated. In contrast, in delayed resolvers, 15-epi-LxA4 and ALX were high early in the response but waned as inflammation progressed. Elevating 15-epi-LxA4 in early resolvers using aspirin increased blister leukocyte ALX but reduced cytokines/chemokines as well as polymorphonuclear leukocyte and macrophage numbers. These findings show that two phenotypes exist in humans with respect to inflammation severity/longevity controlled by proresolution mediators, namely 15-epi-LxA4. These data have implications for understanding the etiology of chronic inflammation and future directions in antiinflammatory therapy.

Novel biphasic role for lymphocytes revealed during resolving inflammation

Acute inflammation is traditionally described as the influx of polymorphonuclear leukocytes (PMNs) followed by monocyte-derived macrophages, leading to resolution. This is a classic view, and despite subpopulations of lymphocytes possessing innate immune-regulatory properties, seldom is their role in acute inflammation and its resolution discussed. To redress this we show, using lymphocyte-deficient RAG1−/− mice, that peritoneal T/B lymphocytes control PMN trafficking by regulating cytokine synthesis. Once inflammation ensues in normal mice, lymphocytes disappear in response to DP1 receptor activation by prostaglandin D2. However, upon resolution, lymphocytes repopulate the cavity comprising B1, natural killer (NK), γ/δ T, CD4+/CD25+, and B2 cells. Repopulating lymphocytes are dispensable for resolution, as inflammation in RAG1−/− and wild-type mice resolve uniformly. However, repopulating lymphocytes are critical for modulating responses to superinfection. Thus, in chronic granulomatous disease using gp91phox−/− mice, not only is resolution delayed compared with wild-type, but there is a failure of lymphocyte re-appearance predisposing to exaggerated immune responses upon secondary challenge that is rescued by resolution-phase lymphocytes. In conclusion, as lymphocyte repopulation is also evident in human peritonitis, we hereby describe a transition in T/B cells from acute inflammation to resolution, with a central role in modulating the severity of early onset and orchestrating responses to secondary infection.

The TGFβ1-Induced Fibronectin in Human Renal Proximal Tubular Epithelial Cells Is p38 MAP Kinase Dependent and Smad Independent

Background/Aim: Transforming growth factor beta 1 (TGFβ1) is a fibrokine implicated in the progression of renal fibrosis. Following TGFβ1 receptor activation, a number of signalling pathways are stimulated. This study investigates the role of p38 mitogen-activated protein (MAP) kinase and Smad pathways in the TGFβ1-induced fibronectin (FN) production. Methods: Transformed human proximal tubular epithelial cells of the line HKC were used. Secreted FN was analyzed by enzyme-linked immunosorbent assay and Smad proteins by Western blotting. Chemical inhibitors were used to study the role of p38 MAP kinase and the TGFβ receptor ALK5. The Smad pathway was studied using a cell line overexpressing Smad7 and small interfering RNAs (siRNA). The FN mRNA expression was assessed by reverse transcription-polymerase chain reaction. Results: TGFβ1 produced a significant increase in FN secretion in both HKC and Smad7-HKC cells, and the p38 MAP kinase inhibitor SB202190 markedly reduced this (n = 3, p < 0.05 and p < 0.01). ALK5 inhibition also reduced the TGFβ1-induced FN (n = 3, p < 0.05). Smad knockdown using the siRNA did not reduce the TGFβ1-induced FN secretion. TGFβ1 induced FN mRNA expression in HKC cells, and SB202190 decreased this induction (n = 5, p < 0.05). Conclusions: These results suggest that TGFβ1-induced FN production in HKC cells is p38 MAP kinase dependent and Smad independent. Targeting p38 MAP kinase may be of therapeutic value in renal fibrosis.

Modelling angiogenesis in inflammation

Angiogenesis is an integral component of chronic inflammatory lesions and is essential for tissue development and repair. The inhibition of this process is a target for the development of novel therapeutics against chronic inflammation, especially those diseases where angiogenic blood vessels feature prominently, such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and psoriasis. The development of these vessels is stimulated by factors produced within the inflammatory milieu and are derived from inflammatory cells, not least macrophages, which produce angiogenic factors under the hypoxic conditions found within these tissues. Macrophages for example have the extraordinary capacity to produce just about every angiogenic growth factor and cytokine known [1, 2, 3], such as tumour necrosis factor (TNF)-α, basic fibroblast growth factor (FGF-2), transforming growth factor (TGF)-β, angiotropin, prostaglandin (PG) E2, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, platelet-derived growth factor (PDGF), interleukin (IL)-6, vascular endothelial growth factor (VEGF), nitric oxide (NO), and angiotensin converting enzyme (ACE), but not all, angiogenin and platelet factor (PF) 4 being absent from their armamentarium. They do synthesise thrombospondin-1 (TP-1), which may be angiogenic or angiostatic depending on whether it is matrix bound or in the soluble or truncated form [4, 5, 6]. Thus, angiostatic factors may also be synthesised or elaborated, e.g. macrophage-derived enzymes such as metallo-elastase may mediate angiostatin release [7].